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Mechanism of Transport of Ferric Enterobactin through FepA in Gram Negative Bacteria
Author(s) -
Majumdar Aritri,
Trinh Vy,
Moore Kyle J.,
Smallwood Chuck R.,
Newton Salete M.C.,
Klebba Phillip E.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.861.5
Subject(s) - periplasmic space , bacterial outer membrane , chemistry , ligand (biochemistry) , enterobactin , cysteine , transmembrane domain , biophysics , stereochemistry , biochemistry , mutant , crystallography , membrane , escherichia coli , receptor , biology , gene , enzyme
BACKGROUND AND PURPOSE Ligand‐gated porin (LGP) receptors of Gram‐negative bacterial outer membrane (OM), typified by FepA, FhuA, BtuB, possess a complex β‐barrel transmembrane channel occluded by a globular N‐terminal domain. The structure poses immediate questions on the mechanism of ligand transport through the receptor protein. Our previous results with in‐vivo fluorescent labeling of engineered Cysteine residues on FepA indicated a ‘Ball and Chain’ mechanism of transport in which the N‐domain is expelled into the inter‐membrane periplasmic space during Ferric enterobactin (FeEnt) ligand passage. This study further investigates whether the N‐domain exits the barrel as a unit or unfolds into the periplasm or forms a ‘transient pore’ within the barrel allowing ligand passage. METHOD We constructed a series of double‐cysteine mutants located i) in the N‐domain such that disulphide bonds should not interfere with transport if it exits the channel as a unit ii) in the N‐domain and the β‐barrel such that the cysteines can come close and form double bonds only if the N‐domain exists the channel during ligand uptake. We tested these mutants for ligand uptake capabilities in nutrition tests and conventional radiochemical assays in the presence and absence of a reductant (β‐mercaptoethanol) of disulphide bonds. RESULTS AND CONCLUSION Our results suggest that three double‐cysteine mutations G27C/R126C, A33C/E120C and L125C/V141C prevent transport of FeEnt in the absence of β‐mercaptoethanol. The location of these residues on the N‐domain point to a mechanism in which the globular N‐domain undergoes conformational change to allow tight ligand passage. Support or Funding Information 1R21AI1157187 granted to PEK